A STRATEGY TO QUANTIFY PROTEIN STABILITY

量化蛋白质稳定性的策略

• 批准号: 8365801
• 负责人: STANLEY FIELDS
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365801
• 关键词: Affect; Biology; Cells; Chimeric Proteins; Enzymes; Eukaryota; Funding; Fungal Genome; Galactose; Genetic; Glucose; Grant; Libraries; Maps; Molecular Chaperones; Mutation; National Center for Research Resources; Principal Investigator; Proteins; Proteolysis; Research; Research Infrastructure; Resolution; Resources; Series; Signal Transduction; Source; System; Testing; Ubiquitin; United States National Institutes of Health; Yeasts; base; cost; design; insight; multicatalytic endopeptidase complex; mutant; promoter; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ubiquitin proteasome system (UPS) governs most of the regulated proteolysis in eukaryotes. Substrates destined for proteasomal degradation are often modified with ubiquitin. The ubiquitin is attached to these proteins by a series of enzymes called E1, E2, and E3. A degron is a primary degradation signal of UPS substrates that is recognized by E3 enzymes or chaperones. We have designed a high-resolution strategy to map the sequence-function relationship of known degrons and to discover degrons in a systematic and high throughput manner. We will combine simple genetic tools with high-throughput sequencing to characterize the degradation signal. Our system is based on the fact that yeast cells that express Ura3 die in the presence of 5-FOA because Ura3 converts 5-FOA into a toxic product. We can alter the stability of Ura3 by fusing it to degrons, which can affect the sensitivity of yeast to 5-FOA. We are currently optimizing this system using the well-characterized degradation signal Deg1 from Matα2 fused to Ura3. This fusion protein is under the control of galactose-regulatable promoter. After the expression of the Ura3-degron fusion is shut off by addition of glucose to the media, the stability of the Ura3-degron fusion in the cells is determined by testing how sensitive the cells are to 5-FOA. We plan to generate a library of mutant Deg1 degrons fused to Ura3 to determine the effect of mutations in the degron for their ability to destabilize Ura3. Cells that harbor functional Ura3-degron fusions will degrade the Ura3 and grow in media containing 5-FOA, and cells that express non-functional degrons will be lost. By comparing the sequences of degrons in the selected yeast to the input culture we will gain insight into how mutations affect the stability of Ura3.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 泛素蛋白酶体系统（UPS）控制着真核生物中的大多数调节蛋白水解。原定用于蛋白酶体降解的底物通常用泛素修饰。 泛素通过一系列称为E1，E2和E3的酶连接到这些蛋白质上。 DEGRON是UPS底物的主要降解信号，由E3酶或伴侣蛋白识别。我们设计了一种高分辨率策略，以绘制已知Degrons的序列功能关系，并以系统和高通量的方式发现DEGRON。 我们将将简单的遗传工具与高通量测序结合在一起，以表征降解信号。我们的系统基于这样一个事实，即表达URA3在5-FOA存在下死亡的酵母细胞，因为URA3将5-FOA转化为有毒产品。我们可以通过将其融合到降解中来改变URA3的稳定性，从而影响酵母对5-FOA的敏感性。 目前，我们正在使用从MATα2融合到URA3的特征良好的降解信号DEG1来优化该系统。 该融合蛋白在半乳糖调节启动子的控制之下。 通过向培养基中添加葡萄糖，将URA3-DEGRON融合的表达关闭后，通过测试细胞对5-FOA的敏感性如何确定细胞中URA3-DEGRON融合的稳定性。我们计划生成与URA3融合的突变deg1 degrons库，以确定DeGron中突变的影响，以使其破坏URA3的能力。 具有功能性URA3-DEGRON融合的细胞将降解URA3并在含有5-FOA的培养基中生长，并且表达非功能性降解的细胞将丢失。通过将选定酵母中的Degrons的序列与输入培养物进行比较，我们将深入了解突变如何影响URA3的稳定性。